1. Field of the Invention
The present invention relates to the field of organic compounds and to methods for specifically binding and labeling enzymes, particularly cysteine proteases, and more particularly legumain, also known as asparaginyl endopeptidase. The present compounds may be labeled for use in imaging and are designed to chemically attach to the active site of the target enzyme.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual structures or methods used in the present invention may be described in greater detail in the materials cited below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Cysteine proteases are proteolytic enzymes, which utilize a cysteine residue for their catalytic activity. They can be grouped into at least 30 protein families. Each family contains proteins with similar amino acid sequences and evolutionarily conserved sequence motifs, which are reflected in the family members' similar 3D structures.
Proteases in Family C1 (the papain family) include mammalian enzymes such as cathepsins B and L, which are thought to be involved in cancer growth and metastasis. Cathepsin K is considered to be involved in bone degradation an osteoporosis. Family C1 also includes parasitic enzymes being essential for the parasite-host interaction (e.g., cruzipain from Trypanosoma cruzi—causing Chagas' disease, and falcipain from Plasmodium falciparum—causing malaria). Enzymes belonging to Family C13 (the legumain family) have been shown to play key roles in antigen presentation.
Asparaginyl endopeptidase, or legumain (Enzyme Class 3.4.22.34), is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents.
Legumain is a lysosomal cysteine protease that was named based on its propensity to cleave protein substrates on the C-terminal side of asparagine residues (1). Legumain is expressed in diverse cell types, and in most cases, its functions are unknown. Recently legumain has emerged as an important enzyme in antigen processing (2, 3) and matrix degradation (4, 5) and it is implicated in various pathological conditions including parasite infection (6, 7), atherosclerosis (8) and tumorigenesis (9, 10). For example, legumain is heavily over-expressed in the majority of human solid tumors such as carcinomas of the breast, colon and prostate (9). Furthermore, knock-down of legumain in mouse models of cancer resulted in a marked decrease in tumor growth and metastasis (10). More recently, mice lacking legumain develop disorders similar to hemophagocytic syndrome, a form of hyperinflammatory response (11). Despite the mounting evidence of legumain as a therapeutically important target, especially in tumor progression and metastasis, current methods to study legumain function mainly depend on antibodies and genetic modification, making it difficult to study legumain in its native state.
Small molecule chemical tools such as activity-based probes (ABPs) provide a highly versatile means to monitor protease function and regulation in a wide range of biological systems. Typical ABPs utilize irreversible inhibitors that can covalently modify active site of enzyme in an activity dependent fashion. However, only a few legumain-specific inhibitors have appeared in the literature thus far. All of these inhibitors have a Cbz-Ala-Ala-Asn peptide scaffold that is based on the sequence of a known substrate of legumain (12). In addition, a number of different reactive electrophilic functional groups including aza-Asn halomethylketones (13), aza-Asn epoxides (7) and aza-Asn Michael acceptors (6) have been used to make irreversible legumain inhibitors. Although these inhibitors are highly potent against legumain in vitro, their potency, and more importantly, their selectivity in vivo, has never been tested. We have previously developed a cell-permeable ABP for legumain that is composed of a peptide acyloxymethyl ketone (AOMK) with a PI aspartic acid (14). Although this probe is useful to study active legumain in cells, it has overall poor potency and can readily cross react with caspases, which also optimally bind to aspartic acid containing AOMKs (15). In view of various shortcomings of prior art legumain inhibitors, we decided to create a new class of legumain inhibitors with faster kinetic properties and increased selectivity for legumain for use in vivo imaging studies. We found, among other things, that the new probes described here (LP-1 in particular are) extremely selective toward legumain and exhibit almost no cross-reactivity towards any other enzymes, even in vivo. The results below demonstrate that one sees only labeled legumain in extracts from whole tissues upon administration of the present probes in vivo.
Described below is a new class of aza-Asn ABPs for legumain that are labeled with Cy5 fluorophore, or other label useful for imaging, and also may be tagged with a series of cell-permeabilizing groups. This new generation of legumain probes can have either an epoxide or Michael acceptor warhead and may be used to image active legumain in vivo both in normal tissues and within solid tumors.
Specific Patents and Publications
Epoxides have been used in cysteine protease inhibitors previously. The general epoxide scaffold for JPM-OEt is based on E-64, which was discovered to be a natural product inhibitor of a variety of cysteine protease in 1978 (Hanada, K. et al. Agric. Biol. Chem. 1978 42, 523-528 and 529-536).

A related compound, JPM-565, is identical to JPM-OEt except that the ethyl ester is converted to the free carboxylic acid.
These classes of compounds have been used as cysteine protease inhibitors since 1978. A number of research groups have synthesized analogs of the general epoxide structure over the past 20+ years and the crystal structure of E-64 bound to various cysteine proteases in the cathepsin family were reported as early as 1989.
Greenbaum et al. Chem. Biol. 2002, 9, 1085-1094 also describe cysteine protease inhibitors that target the same subset of proteases targeted by E-64 and JPM-565. It is noted there that the epoxide class of cysteine protease binding compounds has a chiral structure, e.g., as shown. The following general inhibitor is disclosed:

Libraries of different amino acids substituted for P2, P3, and P4 were prepared. The libraries were first prepared by fixing each of the P2, P3, and P4 positions with each of the 20 possible natural amino acids (minus cysteine and methionine, plus norleucine).
It should be noted that these structures do not utilize N—N bonds or N-linked side chains.